Therapeutic agent for soft tissue sarcoma

ABSTRACT

A therapeutic agent for soft tissue sarcoma (particularly synovial sarcoma) contains a histone deacetylase inhibitor (particularly compound of formula I) as an active ingredient

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.10/875,382, filed Jun. 25, 2004, now pending. The entire teachings ofthe above application are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a therapeutic agent for soft tissuesarcoma, which contains a histone deacetylase inhibitor as an activeingredient.

BACKGROUND ART

In general, when there is a report on a substance or a compound havingan antitumor activity and the report is based solely on in vitroresults, it has been pointed out that such reported results do notdirectly suggest in vivo results. In other words, a substance showing anantitumor activity in vitro does not necessarily show an antitumoractivity in vivo, and application of a substance showing an antitumoractivity in vitro directly as an antitumor agent is difficult.

For example, it has been reported that a compound represented by theformula (I)

(hereinafter to be also referred to as compound A; SEQ ID NO: 1),particularly a stereoisomer of the formula (II)

(hereinafter to be also referred to as compound B or FK228), selectivelyinhibits histone deacetylase to derive a potent antitumor activity, andthat this substance causes high acetylation of histone in the treatedcells, thereby inducing transcription-regulatory activity of variousgenes, cell cycle inhibitory activity and apoptosis (e.g., JP-B-7-64872(corresponding to U.S. Pat. No. 4,977,138), “Experimental CellResearch”, US (1998), vol. 241, pp. 126-133). As the situation nowstands, however, there are many problems yet to be solved, such aswhether or not in vitro results are directly applicable in vivo, whetheror not a useful in vivo effect can be afforded in any tumor, and thelike. No report has ever verified in vitro and in vivo antitumoractivities against soft tissue sarcoma (particularly synovial sarcoma)of the present invention.

Histone deacetylase is a metallo-deacetylating enzyme coordinating Zn atan active center (M. S. Finnin et al., Nature, 401, 188-193 (1999)).This enzyme is considered to change affinity of various acetylatedhistones for DNA. The direct biological phenomenon brought thereby is achange in the chromatin structure. The minimum unit of the chromatinstructure is a nucleosome wherein 146 bp DNA is wound 1.8 timesanticlockwjse around a histone octamer (H2A, H2B, H3 and H4, each 2molecules, core histone). The core histone stabilizes the nucleosomestructure by interaction of the positive charge of the N-terminus ofeach histone protein with DNA. Acetylation of histone is controlled bythe equilibrium between an acetylation reaction involving histoneacetyltransferase and a deacetylation reaction involving histonedeacetylase. It is considered that the histone acetylation occurs at alysin residue where the histone protein N-terminus is evolutionallypreserved well, due to which a core histone protein loses charges at theN-terminus, interaction with DNA is attenuated, and the structure ofnucleosome becomes unstable. Accordingly, the histone deacetylation isconsidered to be the reverse thereof, namely, a shift towardstabilization of the nucleosome structure. However, to what degree theacetylation changes the chromatin structure and how it relates to thetranscriptional regulation etc. secondarily induced thereby are unclearin many aspects.

As genetic characteristics of synovial sarcoma, it has been reportedthat, in about 97% of the entire synovial sarcomas, SYT gene present inthe 18th chromosome and SSX gene present on the X chromosome are fuseddue to chromosomal translocation t (18,X) to express a chimera proteincalled SYT-SSX, and SYT protein constituting the N-terminal region ofthis protein is bound with a chromatin remodeling-associated proteinsuch as p300 and BRM to form a complex (Josiane E. Eid et al., Cell,102, 839-848 (2000)). Synovial sarcoma is one kind of soft tissuesarcoma developed in the four limbs and trunk of the body of males andfemales, and its primary therapy includes removal of tumor by operationand chemotherapy before and after the operation. However, chemotherapyis associated with poor prognosis and a five-year survival rate is about60-70%. Thus, an effective cure has not been established as yet.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a therapeutic agent forsoft tissue sarcoma (particularly synovial sarcoma), which contains ahistone deacetylase inhibitor, particularly compound A, compound B, itsreduction products, metabolites, derivatives, prodrugs, and otheranalogs known to have a strong histone deacetylase inhibitory activity,or a pharmaceutically acceptable salt thereof, as an active ingredient.

In an attempt to solve the above-mentioned problems, the presentinventors have considered that, in synovial sarcoma, formation of theaforementioned complex of SYT-SSX protein, a chromatinremodeling-associated protein and histone deacetylase (HDAC)-associatedprotein enhances histone deacetylase activity, which in turn has aneffect on the canceration, development and/or proliferation, of synovialsarcoma, and have conducted intensive studies of the effect of histonedeacetylase inhibition on various synovial sarcoma cell strains(HS-SY-2, YaFuSS, SYO-1) that express SYT-SSX protein. As a result, theyhave found that compound B and tricostatin A, which are histonedeacetylase inhibitors, exhibit a potent antiturnor activity in vitroand in vivo against SYT-SSX protein expressing cells. Furthermore, theyhave found that they also exhibit a potent antitumor activity against asynovial sarcoma cell strain (HTB93; which is the ATCC identificationnumber of cell line SW982) not expressing SYT-SSX protein. Accordingly,the present invention provides the following.

-   (1) A therapeutic agent for soft tissue sarcoma, which comprises a    histone deacetylase inhibitor as an active ingredient.-   (2) The therapeutic agent of the above-mentioned (1), wherein the    soft tissue sarcoma is synovial sarcoma.-   (3) The therapeutic agent of the above-mentioned (1) or (2), wherein    the soft tissue sarcoma or synovial sarcoma is an SYT-SSX protein    expressing sarcoma.-   (4) The therapeutic agent of the above-mentioned (1), wherein the    histone deacetylase inhibitor is compound A or compound B, or a    reduced form thereof, an analog thereof, a prodrug thereof or a    pharmaceutically acceptable salt thereof.-   (5) The therapeutic agent of the above-mentioned (4), wherein the    soft tissue sarcoma is synovial sarcoma.-   (6) The therapeutic agent of the above-mentioned (5), wherein the    synovial sarcoma is SYT-SSX protein expressing sarcoma.-   (7) A pharmaceutical composition for the treatment of soft tissue    sarcoma, which comprises a histone deacetylase inhibitor and a    pharmaceutically acceptable carrier.-   (8) The pharmaceutical composition of the above-mentioned (7),    wherein the histone deacetylase inhibitor is compound A or compound    B, or a reduced form thereof, an analog thereof, a prodrug thereof    or a pharmaceutically acceptable salt thereof.-   (9) A method for treating soft tissue sarcoma, synovial sarcoma, or    an SYT-SSX protein expressing sarcoma, which comprises administering    an effective amount of a histone deacetylase inhibitor.-   (10) The method of the above-mentioned (9), wherein the histone    deacetylase inhibitor is compound A or compound B, or a reduced form    thereof, an analog thereof, a prodrug thereof or a pharmaceutically    acceptable salt thereof.-   (11) Use of a histone deacetylase inhibitor for the production of a    therapeutic agent for soft tissue sarcoma.-   (12) The use of the above-mentioned (11), wherein the histone    deacetylase inhibitor is compound A or compound B, or a reduced form    thereof, an analog thereof, a prodrug thereof or a pharmaceutically    acceptable salt thereof.-   (13) A commercial package comprising the pharmaceutical composition    of the above-mentioned (7) and a written matter associated therewith    the written matter stating that said pharmaceutical composition can    or should be used for the treatment of soft tissue sarcoma.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is a graph showing an in vitro antitumor action of FK228 againstHS-SY-2 synovial sarcoma cell strain, which is one of the SYT-SSXprotein expressing synovial sarcoma cell strains.

FIG. 2 is a graph showing an in vitro antitumor action of FK228 againstYaFuSS synovial sarcoma cell strain, which is one of the SYT-SSX proteinexpressing synovial sarcoma cell strains.

FIG. 3 is a graph showing an in vitro antitumor action of FK228 againstSYO-1 synovial sarcoma cell strain, which is one of the SYT-SSX proteinexpressing synovial sarcoma cell strains.

FIG. 4 is a graph showing an in vivo antitumor action of FK228 againstSYO-1 synovial sarcoma cell strain, which is one of the SYT-SSX proteinexpressing synovial sarcoma cell strains.

FIG. 5 is a graph showing an in vitro antitumor effect of FK228 on HTB93synovial sarcoma cell strain, which is one of the synovial sarcoma cellstrains that do not express SYT-SSX protein.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The “histone deacetylase inhibitor”, also referred to as “HDACinhibitor” or “HDACi”, in the present invention is a compound that bindsto an active site of histone deacetylase competitively with substrates,and/or a compound that reduces or inhibits the enzyme activity ofhistone deacetylase, and includes any compound (whether synthetic ornatural) reported or will be reported in the future to have a histonedeacetylase inhibitory activity. To be specific, the aforementionedcompound A, a salt thereof and a derivative thereof (e.g., acetylatedcompound A, thiol form (reduced form) with reduced S-S bond as describedin WO02/06307, analogs thereof (e.g., compounds described in U.S. Pat.No. 6,403,555 etc.), prodrugs thereof, etc.) can be mentioned. Inaddition, Trichostatin A, sodium butyrate, suberoylanilide hydroxamicacid (SAHA), MS-275, cyclic hydroxainic-acid-containing peptide,Apicidin, Trapoxin and the like are the compounds reported to have ahistone deacetylase inhibitory activity.

While compound A (and other HDACi's) may have a stereoisomer (e.g.,compound B) based on an asymmetric carbon atom or a double bond, such asan optically active form, a geometric isomer and the like, all theseisomers and mixtures thereof are also encompassed in the scope of thehistone deacetylase inhibitor to be used in the present invention.

In the present specification, unless particularly specified, a simplereference to compound A means a group of compounds regardless ofstereoisomerism, which include a compound B represented by the formula(II).

Moreover, solvate compounds (e.g., inclusion compounds (e.g., hydrateetc.)), anhydrous forms, other crystal polymorphs and pharmaceuticallyacceptable salts thereof of HDACi's, such as compound A, compound B andsalts thereof, are also encompassed in the scope of the presentinvention.

The compound A or a salt thereof are known and available substances. Forexample, compound B, which is one of the stereoisomers of compound A,can be obtained by culturing a strain belonging to the genusChromobacterium, which is capable of producing compound B, under aerobicconditions, and harvesting the substance from its culture broth. As thestrain belonging to the genus Chromobacterium, which is capable ofproducing compound B, for example, Chromobacterium violaceum WB968 (FERMBP-1968) can be mentioned. More specifically, compound B can be obtainedfrom a compound B producing strain as described in JP-B-7-64872(corresponding to U.S. Pat. No. 4,977,138). The compound B is preferablyharvested from a strain belonging to the genus Chromobacterium, which iscapable of producing compound B, because it can be obtained more easily.Synthetic or semi-synthetic compound B is also advantageous in thatfurther purification step is not necessary or the number of steps can bereduced. Similarly, compounds A other than compound B can be alsoobtained by semi-synthesis or total synthesis by conventionally knownmethods. To be more specific, it can be produced according to the methodreported by Khan W. Li, et al. (J. Am. Chem. Soc., Vol. 118, 7237-7238(1996)).

A pharmaceutically acceptable salt of HDACi's, such as the salt ofcompound A or compound B, includes salts with a base or an acid additionsalt such as salts with inorganic base (e.g., alkali metal salts such assodium salt, potassium salt etc., alkaline earth metal salts such ascalcium salt, magnesium salt etc., ammonium salt), salts with an organicbase (e.g., organic amine salts such as triethylamine salt,diisopropylethylamine salt, pyridine salt, picoline salt, ethanolaminesalt, triethanolamine salt, dicyclohexylaxnine salt,N,N′-dibenzylethylenediamine salt etc.), inorganic acid addition salts(e.g., hydrochloride, hydrobromide, sulfate, phosphate etc.), organiccarboxylic acid or sulfonic acid addition salts (e.g., formate, acetate,trifluoroacetate, maleate, tartrate, fumarate, methanesulfonate,benzenesulfonate, toluenesulfonate etc.), salts with a basic or acidicamino acid (e.g., arginine, aspartic acid, glutamic acid etc.) and thelike.

In the present invention, in vivo and in vitro mean as they aregenerally used in this field. Namely, “in vivo ” means a state wherefunctions and reactions of the target living organism can be expressedin living organisms, and in vitro” means that such functions andreactions can be expressed in vitro (tissue culture system, cell culturesystem, cell-free system etc.).

Soft tissue sarcomas include malignant fibrous histocytoma, liposarcoma,rhabdomyosarcoma, leiomyosarcoma, synovial sarcoma, fibrosarcoma,malignant schwannoma, angiosarcoma, clear cell sarcoma and the like.

In addition, gene diagnosis of SYT-SSX protein expressing synovialsarcoma enables selection of patients before treatment, for whom thehistone deacetylase inhibitor of the present invention proves effective.

The therapeutic agent for soft tissue sarcoma of the present inventioncan be used in the form of a pharmaceutical preparation such as a solid,semisolid or liquid preparation (tablet, pellet, troche, capsule,suppository, cream, ointment, aerosol, powder, liquid, emulsion,suspension, syrup, injection etc.) containing a histone deacetylaseinhibitor as an active ingredient, which is suitable for transrectal,intranasal, pulmonary, vaginal, external (topical), oral or parenteral(including subcutaneous, implantation, intravenous and intramuscular)administration.

The therapeutic agent for soft tissue sarcoma of the present inventioncan be also produced by conventional methods using various organic orinorganic carriers conventionally used for forming pharmaceuticalpreparations, such as excipients (e.g., sucrose, starch, mannitol,sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calciumcarbonate etc.), condensation agents (e.g., cellulose, methyl cellulose,hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic,polyethylene glycol, sucrose, starch etc.), disintegrants (e.g., starch,carboxymethyl cellulose, carboxymethyl cellulose calcium, hydroxypropylstarch, sodium starch glycolate, sodium hydrogen carbonate, calciumphosphate, calcium citrate etc.), lubricants (e.g., magnesium stearate,aerosil, talc, sodium lauryl sulfate etc.), corrigents (e.g., citricacid, menthol, glycine, orange powder etc.), preservatives (e.g., sodiumbenzoate, sodium hydrogen sulfite, methylparaben, propylparaben etc.),stabilizers (citric acid, sodium citrate, acetic acid etc.), suspensions(e.g., methyl cellulose, polyvinyl pyrrolidone, aluminum stearate etc.),dispersants (e.g., hydroxypropylmethyl cellulose etc.), diluents (e.g.,water etc.), wax base materials (e.g., cacao butter, polyethyleneglycol, white petrolatum etc.) and the like.

While the administration method of the therapeutic agent for soft tissuesarcoma of the present invention is not particularly limited,intravenous, intramuscular or oral administration is preferable. Inaddition, while a therapeutically effective amount of HDACi's, such as,compound A or compound B or a pharmaceutically acceptable salt thereof,when it is used for a human as an active ingredient varies depending onthe age and condition of individual patient to be treated, and the kindof soft tissue sarcoma, in the case of an intravenous administration,the daily dose of compound A and compound B is generally 0.1-100 mg,preferably 1-50 mg, more preferably 5-30 mg, in the amount of compoundA, per 1 m² of human body surface area, which is given for the treatmentof sarcoma by continuous infusion.

Furthermore, the HDAC1's in the present invention can be administeredalone or in combination with an additional anti-tumor treatment, such assurgery, radiation therapy and/or chemotherapy. Examples ofchemotherapeutic agents include DNA cross-linkers, alkylating antitumoragents, antimetabolite antitumors, and taxanes. Preferredchemotherapeutic agents include cipslatin, 5-fluorouracil,paclitaxel(taxol), docetaxel, and the like.

EXAMPLES

The present invention is specifically explained in detail in thefollowing by referring to Examples, which are not to be construed aslimitative.

Example 1

An SYT-SSX protein expressing synovial sarcoma cell line HS-SY-2(established and kindly provided by Dr. Hiroshi Sonobe, Department ofPathology, National Fukuyama Hospital), YaFuSS (established and kindlyprovided by Dr. Junya Toguchida, Department of Tissue Regeneration,Institute for Frontier Medical Sciences, Kyoto University) and SYO-1(established and kindly provided by Dr. Akira Kawai, Department ofOrthopedics, Faculty of Medicine, Okayama University (now Department ofOrthopedics, National Cancer Center)) were cultured in DMEM (Dulbecco'smodified Eagle's medium) containing 10% (v/v) fetal bovine serum (FBS),100 U/ml penicillin and 100 μg/ml streptomycin at 37° C. under 5% CO₂environment. These cells were plated and cultured for 24 hr, detachedwith 0.25% (w/v) trypsin and recovered. For cell growth ability, an MTTanalysis kit (Colorimetric (MTT) assay for cell survival andproliferation kit of CHEMICON International, Inc.) was used. Each cellstrain was plated in a 96 well microtiter plate at 10³ cell/well, andafter culture for 24 hr, exposed to a 0.1% (v/v) dilute ethanol solutionof FK228 at a concentration distribution of 0.1 nM, 0.2 nM, 1 nM, 50 nMand 100 nM, and 0.1% (v/v) ethanol (Et-OH 0.1% in FIG. 1) as a control.After exposure for 24 hr, 48 hr and 96 hr, each culture was passedthrough a 570 nM filter and the absorbance was measured. All wereperformed with n=4.

The results are shown in FIG. 1, FIG. 2 and FIG. 3. FK228 showed an invitro antitumor effect on SYT-SSX protein expressing synovial sarcoma, asoft tissue sarcoma.

Example 2

Inbred male (BALB/C/nu/nu) nude mice were supplied by Charles RiverJapan, Inc. The animals were all fed and handled according to the AnimalTest Guideline, Animal Resources Division, Advanced Science ResearchCenter, Okayama University. FK228 was administered after 10 days fromsubcutaneous implantation of 10⁵ cells each of the SYO-1 cell strain.The tumor volume was assumed by measuring two diameters perpendicular toeach other using calipers and from the following formula (tumorvolume=⅙π [(d1×d2)^(3/2)] (wherein d1 and d2 are two perpendiculardiameters)). The dose was evaluated by intravenously administering adilute FK228 solution (50 μl, 10% HCO60 which is polyoxyethylene (60)hydrogenated castor oil, diluted with physiological saline) at 0 mg/kg,1.6 mg/kg or 3.2 mg/kg to 20 animals, and as a control, a 3.2 mg/kgdilute FK228 solution was intravenously injected to 7 animals free oftumor implantation. The administration was performed 3 times every 4days, the tumor volume was also measured every 4 days, as well as aftercompletion of the administration.

The results are shown in FIG. 4, wherein the measurement days are shownin terms of the number of days after the subcutaneous implantation.FK228 showed an in vivo antitumor effect on SYT-SSX protein expressingsynovial sarcoma, a soft tissue sarcoma.

Example 3

An SYT-SSX protein non-expressing synovial sarcoma cell line HTB93(purchased from ATCC: American Type Culture Collection) was cultured inDMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) fetalbovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at37° C. under 5% CO₂ environment. These cells were plated and culturedfor 24 hr, detached with 0.25% (w/v) trypsin and recovered. For cellgrowth ability, an MTT analysis kit (Colorimetric MTT) assay for cellsurvival and proliferation kit of CHEMICON International, Inc.) wasused. Each cell strain was plated in a 96 well microtiter plate at 2×10³cell/well, and after culture for 24 hr, exposed to a 0.1% (v/v) diluteethanol solution of FK228 at a concentration distribution of 0.001 nM,0.01 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM and 100 nM, 0.1% (v/v)ethanol as a control and the medium alone as a blank. After exposure for24 hr, 48 hr, 72 hr and 96 hr, each culture was passed through a 570 nMfilter and the absorbance was measured. All were performed with n=4.

For the results, average values of the FK228 addition sample, controland blank were taken, and using numerical values obtained by subtractinga blank value from the value of the FK228 addition sample or control, apercentage corresponding to the ratio of the numerical value of theFK228 addition sample relative to that of the control was taken assurvival rate (%). The results are shown in FIG. 5. FK228 showed an invitro antitumor effect also on SYT-SSX protein non-expressing synovialsarcoma, which is one kind of soft tissue sarcoma.

Sequence Listing Free Text

-   SEQ ID NO: 1: Xaa is an amino acid represented by the formula    NH₂C(CHCH₃)COOH.

In the formula COOHCH₂CH(CHCHC₂H₄SH)OH, the carboxylic group is bondedwith the amino group of the first amino acid Val, the hydroxyl group isbonded with the carboxylic group of the fourth amino acid Val, and theSH group is bonded with the SH group of the second amino acid Cys via adisulfide bond.

INDUSTRIAL APPLICABILITY

The therapeutic agent for soft tissue sarcoma of the present invention,which contains a histone deacetylase inhibitor (particularly FK228) asan active ingredient, has a superior antitumor action not only in vitrobut also in vivo. Accordingly, it can be clinically used, particularlypreferably for the treatment of soft tissue sarcoma.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. All patents and patent publicationand other publications identified or referenced herein are incorporatedby reference in their entirety.

This application is based on and claims the benefit of patentapplication No. 183643/2003 filed in Japan, the contents of which arehereby incorporated by reference.

1-8. (canceled)
 9. A method for treating soft tissue sarcoma, whichcomprises administering an effective amount of a histone deacetylaseinhibitor.
 10. The method of claim 9, wherein the soft tissue sarcoma issynovial sarcoma.
 11. The method of claim 10, wherein the synovialsarcoma is SYT-SSX protein expressing sarcoma.
 12. The method of claim9, wherein the soft tissue sarcoma is selected from the group consistingof malignant fibrous histocytoma, liposarcoma, rhabdomyosarcoma,leiomyosarcoma, synovial sarcoma, fibrosarcoma, malignant schwannoma,angiosarcoma, and clear cell sarcoma. 13-16. (canceled)
 17. The methodof claim 9, wherein the histone deacetylase inhibitor is SAHA ortrichostatin A.
 18. The method of claim 17, wherein the soft tissuesarcoma is synovial sarcoma.
 19. The method of claim 18, wherein thesynovial sarcoma is SYT-SSX protein expressing sarcoma.
 20. The methodof claim 17, wherein the soft tissue sarcoma is selected from the groupconsisting of malignant fibrous histocytoma, liposarcoma,rhabdomyosarcoma, leiomyosarcoma, synovial sarcoma, fibrosarcoma,malignant schwannoma, angiosarcoma, and clear cell sarcoma. 21-23.(canceled)